A new, general method for lipid extraction and measurement of vitamin E/total lipid ratios in tissue and cell samples has been developed. The new extraction procedure uses a combination of sodium dodecylsulfate, ethanol and n-heptane, and is mild, clean, convenient, efficient and rapid (less than or equal to 5 min). The efficiency of the new method has been confirmed for human plasma, red blood cells and rat liver homogenate by the comparison of the yields of vitamin E, O-acyl lipid and cholesterol with the yields obtained following conventional extraction procedures. Extraction efficiency also has been confirmed for multilamellar vesicles composed of known quantities of vitamin E, egg lecithin and cholesterol.
The net rates of uptake of the natural (2R,4'R,8'R) diastereoisomer of alpha-tocopherol (alpha-T) and the biodiscrimination relative to its 2S-epimer (2S,4'R,8'R) have been measured, in two experiments, for the blood and 21 tissues of male Sprague-Dawley rats fed over a period of several months diets containing deuterium-substituted forms of the alpha-T acetates. Gas chromatography-mass spectrometry was used to measure the amount of deuterated tocopherols taken up relative to the amount of nondeuterated tocopherol remaining. The measurements were performed at different times after the rats, placed for one month on a basal diet containing nondeuterated, natural alpha-T acetate, were switched to a diet containing the same total quantity of deuterated forms of either natural alpha-T acetate or a mixture of the acetates of the 2R- and 2S-epimers (i.e., ambo-alpha-T acetate). In experiment 1 the source of vitamin E in the replacement diet was trideuterio-2R,4'R,8'R-alpha-T acetate. The data obtained provide the first direct measure of the rate at which natural vitamin E is replaced and augmented in the tissues of growing animals under normal laboratory dietary conditions. There are dramatic differences in the tissue kinetics; for example, the apparent half-life of vitamin E, i.e., the time at which the total amount of ingested trideuterio-alpha-T taken up is the same as the amount of nondeuterated alpha-T remaining, varies from ca. 1 wk for the lung to ca. 11 wk for the spinal cord. In experiment 2 the vitamin E in the replacement diet was an equimolar mixture of trideuterio-2S,4'R,8'R- and hexadeuterio-2R,4'R,8'R-alpha-T acetates. The results show that there is a preferential uptake of the natural diastereoisomer of alpha-T by all tissues (except the liver during the first month). Examination of fecal material reveals that the biodiscrimination begins in the gut; the incomplete hydrolysis of the acetates shows clearly that this reaction proceeds to a greater extent with the natural diastereoisomer. The greatest discrimination of all the tissues examined was found to occur in the brain. After five months, the level of the deuterated natural diastereoisomer was more than five times that of the deuterated 2S-epimer. These results have potential implications for human nutrition.
A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.
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